A central question in the area of steroid hormone action in eukaryotic cells is the nature of the intracellular receptor proteins which are involved in the response of the target tissue to the hormone. Thus, there is a controversy as to the molecular structure of these proteins. The three most likely hypotheses are that the receptor (1) is a single polypeptide chain, (2) is composed of identical or different subunits, or (3) yields different forms due to a specific, limited proteolysis of the receptor. The proposed studies will analyze the biochemical structure of the glucocorticoid receptor protein(s) in mouse cells with these specific hypotheses in mind. Although some studies will utilize mouse liver, the majority will involve the use of a murine lymphoma cell line (WEHI-7) and various receptor mutants obtained from this line. This cell line has an apparently normal diploid complement of receptors (30,000 receptors per cell), whereas previous studies on receptor structure utilized the S49 cell line, which is functionally haploid for the receptor gene (15,000 receptors per cell). This raises a question about the conclusion drawn from these studies that the glucocorticoid receptor is a single molecule. The studies proposed here will involve a critical, thorough analysis of glucocorticoid receptor structure in the WEHI-7 cell line, which appears to be an excellent endocrine model system. Characterization will include the behavior of the receptor on ion-exchange (DEAE-cellulose, phosphocellulose), adsorption (DNA-cellulose), and gel filtration chromatography. Isoelectric focusing will also be used to determine if multiple molecular forms of the receptor exists. Most importantly, mutants will be generated which display an altered amount of receptor or receptors which display altered physicochemical properties. The use of these mutant clones will allow a determination of which of the hypotheses dealing with receptor structure is correct. Studies are also planned to determine if limited proteolysis of the receptor appears to play a physiologically important role in the response of the cell to the hormone. Finally, preliminary experiments are planned to attempt the purification of the WEHI-7 cell line glucocorticoid receptor using a technique which seems to have a general applicability to steroid receptors.